I. Serine Proteases
Serine proteases (E.C. 3.4.21) are the sub-sub class of endopeptidases that use serine as the nucleophile in peptide bond cleavage (Barrett, A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J. et al, Elsevier, Amsterdam, pages 3-22 (1986); and Hartley, B. S., Ann. Rev. Biochem., 29:45-72 (1960)).
Serine proteases are well known in the art and two superfamilies of serine proteases, i.e., the chymotrypsin superfamily and the Streptomyces subtilisin superfamily, have been observed to date (Barrett, A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J., et al, Elsevier, Amsterdam, pages 3-22 (1986); and James, M. N. G., In: Proteolysis and Physiological Regulation, Ed. Ribbons, D. W. et al, Academic Press, New York, pages 125-142 (1976)).
Examples of serine proteases of the chymotrypsin superfamily include tissue-type plasminogen activator (hereinafter "t-PA"), trypsin, trypsin-like protease, chymotrypsin, plasmin, elastase, urokinase (or urinary-type plasminogen activator (hereinafter "u-PA")), acrosin, activated protein C, Cl esterase, cathepsin G. chymase and proteases of the blood coagulation cascade including kallikrein, thrombin, and Factors VIIa, IXa, Xa, XIa and XIIa (Barrett, A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J., et al, Elsevier, Amsterdam, pages 3-22 (1986); Strassburger, W. et al, FEBS Lett., 157:219-223 (1983); Dayhoff, M.o., Atlas of Protein Sequence and Structure, Vol. 5, National Biomedical Research Foundation, Silver Spring, Md. (1972); and Rosenberg, R. D. et al, Hosp. Prac., 21:131-137 (1986)).
Some of the serine proteases of the chymotrypsin superfamily, including t-PA, plasmin, u-PA and the proteases of the blood coagulation cascade, are large molecules that contain, in addition to the serine protease catalytic domain, other structural domains responsible in part for regulation of their activity (Barrett, A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J., et al, Elsevier, Amsterdam, pages 3-22 (1986); Gerard, R. D. et al, Mol. Biol. Med., 3:449-457 (1986); and Blasi, F. et al, In: Human Genes and Diseases, Ed. Blasi, F., John Wiley & Sons, Ltd., pages 377-414 (1986)).
The catalytic domains of all of the serine proteases of the chymotrypsin superfamily have both sequence homology and structural homology. The sequence homology includes the total conservation of:
(i) the characteristic active site residues (e.g., Ser.sub.195, His.sub.57 and Asp.sub.102 in the case of trypsin); PA1 (ii) the oxyanion hole (e.g., Gly.sub.193, Asp.sub.194 in the case of trypsin); and PA1 (iii) the cysteine residues that form disulfide bridges in the structure (Hartley, B. S., Symp. Soc. Gen. Microbiol., 24:152-182 (1974)). PA1 (i) the common fold that consists of two Greek key structures (Richardson, J., Adv. Prot. Chem., 34:167-339 (1981)); PA1 (iii) detailed preservation of the structure within the core of the molecule (Stroud, R. M., Sci. Am., 231:24-88 (1974)).
The structural homology includes:
(ii) a common disposition of catalytic residues; and
A comparison of the sequences of the members of the chymotrypsin superfamily reveals the presence of insertions or deletions of amino acids within the catalytic domains (see for example, FIG. 1). In all cases, these insertions or deletions map to the surface of the folded molecule and thus do not effect the basic structure of the molecule (Strassburger, W. et al, FEBS Lett., 157:219-223 (1983)).